In vivo, cell-cell adhesion plays an important role in a wide range of events including morphogenesis and organ formation, leukocyte extravasion, tumor metastasis and invasion, and the formation of cell junctions. Additionally, cell-cell adhesion is crucial for the maintenance of tissue integrity, e.g., of the intestinal epithelial barrier, of the blood brain barrier and of cardiac muscle.
Intercellular adhesion is mediated by specific cell adhesion molecules. Cell adhesion molecules have been classified into at least three superfamilies including the immunoglobulin (Ig) superfamily, the integrin superfamily and the cadherin superfamily. All cell types that form solid tissues express some members of the cadherin superfamily suggesting that cadherins are involved in selective adhesion of most cell types.
Cadherins have been generally described as glycosylated integral membrane proteins that have an N-terminal extracellular domain that determines binding specificity (the N-terminal 113 amino acids appear to be directly involved in binding), a hydrophobic membrane-spanning domain and a C-terminal cytoplasmic domain (highly conserved among the members of the superfamily) that interacts with the cytoskeleton through catenins and other cytoskeleton-associated proteins. Some cadherins lack a cytoplasmic domain, however, and appear to function in cell-cell adhesion by a different mechanism than cadherins that do have a cytoplasmic domain. The cytoplasmic domain is required for the binding function of the extracellular domain in cadherins that do have a cytoplasmic domain. Binding between members of the cadherin family expressed on different cells is mainly homophilic (i.e., a member of the cadherin family binds to cadherins of its own or a closely related subclass) and Ca.sup.2+ -dependent. For recent reviews on cadherins, see Takeichi, Annu. Rev. Biochem., 59:237-252 (1990) and Takeichi, Science, 251, 1451-1455 (1991).
The first cadherins to be described (E-cadherin in mouse epithelial cells, L-CAM in avian liver, uvomorulin in the mouse blastocyst, and CAM 120/80 in human epithelial cells) were identified by their involvment in Ca.sup.2+ -dependent cell adhesion and by their unique immunological characteristics and tissue localization. With the later immunological identification of N-cadherin, which was found to have a different tissue distribution from E-cadherin, it became apparent that a new family of Ca.sup.2+ -dependent cell-cell adhesion molecules had been discovered.
The molecular cloning of the genes encoding mouse E- [see Nagafuchi et al., Nature, 329: 341-343 (1987)], chicken N- [Hatta et al., J. Cell Biol., 106: 873-881 (1988)], and mouse P-[Nose et al., EMBO J. 6: 3655-3661 (1987)] cadherins provided structural evidence that the cadherins comprised a family of cell adhesion molecules. Cloning of chicken L-CAM [Gallin et al., Proc. Natl. Acad. Sci. USA, 84: 2808-2812 (1987)] and mouse uvomorulin [Ringwald et al., EMBO J., 6: 3647-3653 (1987)] revealed that they were identical to E-cadherin. Comparisons of the amino acid sequences of E-, N-, and P-cadherins showed a level of amino acid similarity of about 45%-58% among the three subclasses. Liaw et al., EMBO J., 9: 2701-2708 (1990) describes the use of PCR with degenerate oligonucleotides based on one conserved region of E-, N- and P-cadherins to isolate N- and P-cadherin from a bovine microvascular endothelial cell cDNA. The Liaw et al., supra, results implied that there were only E-, N-, and P-cadherins because no new cadherins were identified. Also in 1990, it was reported in Heimark et al., J. Cell Biol., 110: 1745-1756 (1990) that an antibody generated to bovine aortic endothelial cells recognized an intercellular junctional molecule designated V-cadherin which had a similar molecular weight to known cadherins and was able to inhibit Ca.sup.2+ -dependent cell endothelial cell adhesion. The article did not disclose any sequence information for the protein recognized by the antibody.
No further cadherin genes were described until the identification of eight of the novel cadherins claimed herein was reported in Suzuki et al., Cell Regulation, 2: 261-270 (1991). Subsequently, several other cadherins were described including chicken R-cadherin [Inuzuka et al., Neuron, 7: 69-79 (1991)], mouse M-cadherin [Donalies et al., Proc. Natl. Acad. Sci. USA, 88: 8024-8028 (1991)], chicken B-cadherin [Napolitano et al., J. Cell. Biol., 113: 893-905 (1991)], and T-cadherin [chicken in Ranscht et al., Neuron, 7: 391-402 (1991) and chicken and human in Patent Cooperation Treaty (PCT) International Publication No. WO 92/08731 published on May 29, 1992].
The determination of the tissue expression of the various cadherins reveals that each subclass of cadherins has a unique tissue distribution pattern. For example, E-cadherin is found in epithelial tissues while N-cadherin is found in nonepithelial tissues such as neural and muscle tissue. The unique expression pattern of the different cadherins is particularly significant when the role each subclass of cadherins may play in vivo in normal events (e.g., the maintenance of the intestinal epithelial barrier) and in abnormal events (e.g., tumor metastatis or inflammation) is considered. Supression of cadherin function has been implicated in the progression of various cancers. See Shimoyama et al., Cancer Res., 52: 5770-5774 (1992). Different subclasses or combinations of subclasses of cadherins are likely to be responsible for different cell-cell adhesion events in which therapeutic detection and/or intervention may be desirable. Studies have also suggested that cadherins may have some regulatory activity in addition to adhesive activity. Matsunaga et al., Nature, 334, 62-64 (1988) reports that N-cadherin has neurite outgrowth promoting activity and Mahoney et at., Cell, 67, 853-868 (1991) reports that the Drosophila fat tumor supressor gene, another member of the cadherin superfamily, appear to regulate cell growth. Expression of the cytoplasmic domain of N-cadherin without its extracellular domain has been shown in Kintner et al., Cell, 69: 229-236 (1992) to disrupt embryonic cell adhesion and in Fugimori et al., Mol. Biol. Cell, 4: 37-47 (1993) to disrupt epithial cell adhesion. Thus, therapeutic intervention in the regulatory activities of cadherins expressed in specific tissues may also be desirable.
There thus continues to exist a need in the art for the identification and characterization of additional cadherins participating in cell-cell adhesion and/or regulatory events. Moreover, to the extent that cadherins might form the basis for the development of therapeutic and diagnostic agents, it is essential that the genes encoding the proteins be cloned. Information about the DNA sequences and amino acid sequences encoding the cadherins would provide for the large scale production of the proteins and for the identification of the cells/tissues naturally producing the proteins, and would permit the preparation of antibody substances or other novel binding molecules specifically reactive with the cadherins that may be useful in modulating the natural ligand/antiligand binding reactions in which the cadherins are involved.